Whole Food Testing

Prof. K. B. Tikoo Head, Dept. of Pharmacology & Toxicology

National Institute of Pharmaceutical Education & Research (NIPER), S.A.S. Nagar.

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FOOD

Food Means any article used as food or drink for

human consumption other than drug and also

includes:

Any article which enters or used in the composition

or preparation of food

Any flavoring matter or condiments

Any other article notified by the Central Govt. e.g.

packaged drinking water

2 Food Safety and Standards Act, 2006, India

“Drugs for the disease and food for the life. Imagine how

good the regulations should be for the thing to which long

term exposure occurs?”

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Drugs & Cosmetics act 1940

CDSCO 1948

134 approved labs+7 central dedicated drug

testing labs

Prevention of Food adulteration act 1954

FSSAI 2011

82 approved+12 central referral food testing labs

Drugs Food

Why Food Safety?...

Globalization of trade in food.

Changing products, processes etc.

Food handling practices

More than 200 known diseases are transmitted

through food1

“Food Safety is a hidden, often overlooked problem”

5 1. Mead et al., (1999). Food-related illness and death in the United States. . Emerg infect dis 5: 607-625.

Advances in Food/Food products

Organic food

Biotechnology-derived (BD) food

Irradiated food

Frozen food

Freeze dried food

Functional foods and Neutraceuticals

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The term „Genetically Modified (GM)‟ foods appears to be a misnomer

because conventional techniques of plant, animal and microbial breeding

also involve genetic modification, which is generally undefined. So, it is apt to use the term „biotechnology-derived

(BD)‟ foods.

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Issues of health effects of BD-foods

Toxicity of transgene per se

• Dietary DNA is not new to our digestive system and that

derived from plant/animals is not toxic, in fact good for gut

function1,2.

• Foreign DNA is removed by hydrolysis during

digestion, excision from the host genome, and targeted

DNA methylation3.

1. FAO/WHO (2000). Safety aspects of genetically modified foods of plant origin: Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology.;

2. Royal Society (2002). Genetically Modified Plants for Food Use and Human Health—An Update. Policy Document 4/02. 3. Doerfler, W. (1991). Patterns of DNA methylation—Evolutionary vestiges of foreign DNA inactivation as a host defense

mechanism. Biol. Chem. Hoppe- Seyler 372: 557–564.

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Toxicity of transgene-product in the whole food

is to be considered on a case-by-case basis

•Transgene products - toxin (eg., Bt endotoxin, which is

harmless in mammals), allergens (eg., Kiwi, which was once

non allergic but now-a-days proved to be allergic).

Examples:

1) StarLink corn: To improve the insecticidal activity of Bt protein,

Cry9C was created. It had negative impact on the health of humans

and hence was not approved1.

2) Nutritional quality of soybeans was sought to be improved using a

brazil nut protein, the methionine rich 2S albumin. It failed because

people allergic to Brazil nuts also reacted to the new soybean2.

1. CDC (2001) Investigation of human health effecs associated with potential exposure to BD corn. A Report to the USFDA from

the CDC

2. Nordlee et al (1996). Identification of a Brazil-nut allergen in transgenic soybeans. N Eng J Med 334: 688–692.

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Unintended effects resulting from the insertion of

transgene into the host genome

Current genetic engineering processes insert transgene

randomly. Therefore, probability of pleiotropic and insertional

mutagenic effects.

Examples:

1) A1 gene codes for salmon red color in Petunia flower. Genetic

modification for improving the yield led to unexpected color

patterns because of deletion of A1 gene/hypermethylation of its

promoter1.

2) Tryptophan-associated eosinophilia-myalgia syndrome (EMS):

When a second BDO was used, the agent responsible for tryptophan-

associated EMS increased in content but no novel toxicant was

created2.

1. Meyer et al. (1992). Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic Petunia and its color phenotype. Molec Gen Genet 231: 345–352.

2. Sullivan et al. (1996). EMS among non-L-tryptophan users and pre-epidemic cases. J Rheumatol 23: 1784–1787

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Transfer of Antibiotic Resistance Marker Genes

from the BD Food to Gut Microbes1

•Genomic DNA is efficiently degraded in the gut

•Plant to microbe gene transfer rate is very low

Effect of transgene-product on non-target

organisms • Bt affects not only the target insects - Lepidoptera, Coleoptera,

or Diptera, but also the larvae of non-target insects like

Monarch butterfly, which helps in pollination2.

1. Royal Society (1998). BD plants for Food use. Policy Document 2/98

2. Losey et al (1999). Transgenic pollen harms monarch larvae. Nature 399: 214.

Misbelief about BD crops

Potential adverse effects of BD varieties on indigenous flora and

fauna brought into the public by anti-BD activists.

Bt gene product affects not only the target insects but also the larvae of

non-target insects like Monarch butterfly, which helps in pollination. This

has been observed at laboratory level but not at field level because of very

limited synchrony between the time point of pollination and formation of

larvae1.

Gatehouse et al. (2002). The case of the monarch butterfly: A verdict is returned. Trends Genet 18: 249–251. 12

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Some academics have aligned with the „consumer

interests‟.

Glyphosate is a broad-spectrum weedicide. Corn was

engineered to be glyphosate-resistant. A study proved its

carcinogenic property. This report was encashed by

many BD activists. But later proved that the study was

methodologically and statistically flawed leading to its

retraction1.

1. Seralini et al. (2012). Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize.

Food Chem Toxicol 63:244

Because of all these, the public now mistrusts most

mainstream sources of data on BD food despite the

endorsements by the regulatory bodies –USFDA,

USNAS, AMA, EC about their safety and the use

of BD crops for decades.

“In terms of risk, how a food crop is created is totally

irrelevant –it is what is in the food that is important”

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Points to be focussed in Safety assessment of BD-foods1:

• The whole food (food product) itself, rather than the process

through which it is made.

• “Substantial Equivalence”

New BD food should be substantially equivalent in composition and

nutritional characteristics to the existing food.

Current safety testing concentrates on the toxicity of single

chemicals rather than the whole food.

No adequate animal models for certain toxic conditions e.g.,

Eosinophilia-Myalgia syndrome (EMS).

Issues of BD Food Testing

1. Hollingworth et al. (2003). The Safety of BD Foods produced through Biotechnology. Toxicol Sci 71:2-8

Whole food (WF) toxicity study design

considerations

If the composition of the food and/or feed derived from BD plant is

substantially modified, or if there are any indications for the

potential occurrence of unintended effects based on the preceding

molecular, compositional or phenotypic analyses, not only new

constituents but also the whole food and feed derived from the BD

plant should be tested.

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Limitations of whole food (WF) animal studies in the

safety assessment of BD crops

Low toxicological power of whole food toxicity studies and flawed

study design and failure resulted in numerous studies of BD crops as

showing adverse effects

eg., Transgenic potato containing snowdrop lectin gene affected

parts of the rat intestine on feeding. Researchers have shown that

some effects are due to lectin per se and the reason behind total

toxicity could not be fully demonstrated.

Limitations of the study

• Potato glycoalkaloids which are toxic to monogastric animals

were not measured.

• Small sample size

• Dietary deficiencies in the rats fed BD potato

Royal Society. (1999). Review of data on possible toxicity of GM potatoes. The Royal Society Ref 11/99. 17

Suitable technique used for studying the safety

eg., Electron microscopic examination of the liver and pancreas

revealed ultra structural anomalies in various studies of mice fed

with 14% BD herbicide-tolerant soybean.

Limitations of the study:

• failure to control for possible litter effects

• inadequate methodological procedures to ensure an unbiased,

quantitative assessment

• inappropriate statistical methods

• differences in the phytoestrogen content of the control and BD

soybean-based feeds

•Electron microscopy of selected tissues is useful to elucidate a

chemical‟s mechanism of action but it is not a recommended approach in

OECD testing guidelines because the relatively small amount of tissue

evaluated cannot be considered representative of the whole organ.

Williams AL, DeSesso JM. (2010). Genetically-modified soybeans – a critical evaluation of studies addressing potential

ultrastructural changes associated with ingestion. Toxicologist 114: 1154. 18

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Whole food toxicity studies utilise inappropriate methods to

detect unintended changes because of lower detection limits.

Defined single substances can be dosed to laboratory animals

at very large multiples of the expected human exposure, thus

giving a large margin of safety. In contrast, foodstuffs are

bulky, lead to satiation and can only be included in the diet at

much lower multiples of expected human intakes.

When testing whole foods, the possible highest concentration

of the BD food and feed in the laboratory animal diet may be

limited because of nutritional imbalance of the diet, or by the

presence of compounds with a known toxicological profile.

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BD food safety testing Inspite of many issues with WF-feeding testing, most commonly used animal

studies of BD food testing are:

42-day broiler chicken study: performance parameters –

mortality/survival, general health, weight gain, feed consumption,

feed conversion, organ weights, etc. are measured. The study

consisted of three 14-d phases (starter, grower, finisher)1.

Sub chronic (90-day) rat study: to detect potential toxicity. Apart

from the parameters of above study, macroscopic and microscopic

(histopathological) findings. Subchronic duration results are

similar to those obtained after chronic exposure2.

1. Herman et al (2009). Performance of broiler chickens fed diets containing DAS-68416-4 soybean meal. GM Crops 2: 169–175.

2. EFSA report, (2008). Safety and nutritional assessment of BD plants and derived food and feed: The role of animal feeding trials.

Food Chem Toxicol 46:S2-70.

Triggers for the conduct of WF toxicity studies

There is no evidence that the insertion of transgene from a non-toxic source

into a BD crop has greater propensity to result in the de novo generation of

novel toxic compounds

Major challenge lies in identifying circumstances under which the WF testing

is scientifically and ethically justified taking into consideration various factors

such as:

a) probability of recombinant DNA technology to produce a BD crop that

will randomly produce novel, unintended toxic substances de novo

b) implausibility of any such substances being highly toxic

c) low concentration of unintended substances likely to be produced

d) inherent limitations of WF toxicity studies

However identifying cases under which WF testing is not needed is easier as

WF toxicity studies are essentially bioassays and are inherently less sensitive

than analytical chemistry techniques

In addition use of a less sensitive WF testing to provide reassurance of the

accuracy of more sensitive methods is difficult to justify. 21

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Public will accept BD foods only when unmet needs are met by them

“Necessity as the mother of acceptance

of BD food”

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In Philippines, vit A deficiency caused blindness, Beta carotene-

enriched Golden Rice was accepted by the people because of its

cheap rate compared to other vitamin A supplements1.

Genetic modification developed disease resistant varieties of

papaya in Hawaii, where the non-BD crop was almost wiped out

by ringspot virus.

BD technology is also used in the orange groves of Florida,

where the harvest is threatened by citrus greening disease2.

1. (2013). Contrary to popular belief. Nat Biotechnol 31:767

2. July 27, 2013. The New York Times

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Knowledge= Prevention